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FENOFIBRATE
Bioanalytical method development and Validation using HPLC
5.1 DRUG PROFILE Fenofibrate
Fenofibrate is an antilipidemic agent which reduces both cholesterol and
triglycerides in the blood.1
Chemistry 2 Molecular Formula : C20H21ClO4 Chemical Name : Propan-2-yl-2-{4-[(4-chlorophenyl) carbonyl]
phenoxy} -2-methylpropanoate
Molecular Weight : 360.831
Description : Fenofibrate occurs as a white or almost white
crystalline powder.
Solubility : It is slightly soluble in ethanol (95%), very soluble in
methylene chloride and practically insoluble in water
Storage : Store below 30°C (86°F) and protect from light.
It is official in British Pharmacopoeia.3
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Mechanism of action
Fenofibrate is an antilipidemic agent, which lowers plasma TG by activating
lipoprotein lipase thus increasing very low density lipoprotein (VLDL) catabolism
with consequent increase in high density lipoprotein (HDL) levels.
Fenofibrate is an antilipidemic agent, which acts by activation of peroxisome
proliferator activated receptor-a (PPARa). Most of the triglyceride-rich particles are
eliminated from plasma by activating lipoprotein lipase and reducing production of
apoprotein C-III due to lipolysis. The resulting decrease in triglycerides produces a
change in the size and composition of LDL from small, dense particles, to big
buoyant particles. These bigger particles have a greater affinity for cholesterol
receptors and are catabolised rapidly.4, 5
Indications
It is indicated for the treatment to reduce elevated LDL-C Total-C,
Triglycerides and Apo B, and to increase HDL-C in adult patients with mixed
dyslipidemia and primary hypercholesterolemia.6
Adverse Effects
Severe stomach pain, Nausea, Vomiting, Unusual weakness or fever, Joint
pain, Indigestion, Bloating, Rash.
Dosage and Administration
The recommended dosage is 200-300mg capsule once daily with food.
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Contraindications 7
1. It is contraindicated in patients who are hypersensitive to the product.
2. Patients with severe hepatic and renal impairment.
3. Patients with Unexplained persistent liver function abnormality and primary
biliary cirrhosis.
4. Pre-existing gall bladder disease.
5. In Pregnancy and lactation.
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5.2: LITERATURE REVIEW: Fenofibrate
Fathy M et al (2011)8 reported two new selective, precise and accurate
methods for the determination of Fenofibrate in the presence of its basic degradation
product. In the first method Fenofibrate was determined using an algorithm bivariate
calibration derivative method. In the second method (HPLC), separation was
performed on RESTEK Pinnacle II phenyl column (5 µm, 250 × 4.6 mm) and
Pinnacle II phenyl (5 µm, 10 × 4 mm) guard cartridge using a mobile phase used
was of methanol-0.1% phosphoric acid (60:40, v/v) at a flow rate 2.0 ml/min, and
the column oven temperature was set at 50°C. The UV detector was time
programmed at 302 nm and 289 nm for the internal standard and Fenofibrate.
Madureira TV et al (2010)9 reported a simple analytical method using
solid-phase extraction followed by a high-performance liquid chromatography with
diode array detection (HPLC-DAD) analysis, compounds included six
pharmaceuticals (Carbamazepine, Diazepam, Fluoxetine, Propranolol,
Sulfamethoxazole and Trimethoprim) and the active metabolite of Fenofibrate.
Briefly, this method consisted of the preconcentration of water samples (2 L) on 500
mg Oasis HLB cartridges and HPLC analysis using a RP18 analytical column in a
gradient mode with a flow rate of 1.0 ml/min. Thus, it is concluded that this method
can be successfully applied for screening pharmaceuticals in polluted estuarine
areas.
Bhavesh D et al (2009)10 reported a rapid, specific and sensitive ultra-
performance liquid chromatography tandem mass spectrometry method for the
determination of Fenofibrate in human plasma. Extraction process was one-step
liquid-liquid extraction coupled with an Acquity UPLC(TM) BEH C18 column (50 x
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2.1 mm, i.d., 1.7 micron) with isocratic elution technique with a flow rate of 0.2
ml/min with Mefenamic acid (internal standard), multiple reaction-monitoring mode
using the electrospray ionization technique.
Korany MA et al (2008)11 reported a differential pulse polarographic and
square wave voltammetric techniques by reduction at a dropping-mercury working
electrode versus Ag/AgCl reference electrode for determination of Etofibrate,
Fenofibrate and Atorvastatin in their pharmaceutical preparations and human plasma
optimum conditions such as pH, scan rate and pulse amplitude were studied.
Kadav AA et al (2008)12 reported a UPLC method for simultaneous
determination of Atorvastatin, Fenofibrate and their impurities in tablets. The
chromatographic separation was carried on acquity UPLC BEH C18 column (1.7
micron, 2.1 mmx100 mm) with gradient elution technique, mobile phase was
acetonitrile and ammonium acetate buffer (pH-4.7; 0.01 M) at flow rate of 0.5
ml/min and detection by UV was at 247 nm. Total run time was 3.0 min within
which main compounds and six other known and major unknown impurities were
separated. All the validation parameters were done for Atorvastatin, Fenofibrate and
their known impurities.
Straka RJ et al (2007)13 reported a RP-HPLC method in combination with
anion-exchange solid-phase extraction, which rapidly and accurately determines
steady-state Fenofibrate serum concentrations. Isocratic conditions were used for
Chromatographic separation by using ultraviolet detection at 285 nm.
Concentrations were found to be linear over the range of 0.5 to 40.0 mg/L for
Fenofibric acid analysis. Accuracies were found from 98.65% to 102.4%.
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Alaa EG et al (2005)14 reported two spectrophotometric and HPLC methods
for the estimation of Fenofibrate, Vinpocetine and their hydrolysis products. The
resolution of drugs and hydrolysed products were estimated by using numerical
spectrophotometric methods such as partial least squares (PLS-1) and principal
component regression (PCR) applied to obtained UV spectra, and graphical methods
as first derivative of ratio spectra (1DD) or first (1D) and second (2D) derivative
spectrophotometry for Vinpocetine and Fenofibrate. HPLC method was developed
using ODS column with mobile phase consisting of acetonitrile-water (80:20 v/v,
pH-4.0) with λmax at 287 nm for Fenofibrate and a mobile phase consisting of
acetonitrile-10 mM KH2PO4, containing 0.1% diethylamine (60:40 v/v, pH 4.6) with
λmax at 270 nm for Vinpocetine.
Shihabi ZK (2004)15 reported capillary electrophoresis method to measure
Fenofibrate in capsules based on micellar electro kinetic capillary chromatography
with UV detection at 280 nm by employing a borate buffer containing sodium
dodecyl sulfate (SDS).
Meikle AW et al (2003)16 reported a simple HPLC-MS/MS method which
was evaluated on two patients who are suffering from Cushing syndrome who had
elevated Urinary free cortisol (UFC) excretion, which is usually done for diagnosis
of Cushing syndrome. Regular laboratory testing was inconsistent with the diagnosis
of Cushing syndrome which raised doubts. By identifying a probable cause of
analytical interference from Fenofibrate (Tricor), medication taken by the patients.
Fenofibrate peak was overlapped with the HPLC peak of cortisol and produced an
MS/MS transition overlapping the major transition of cortisol. HPLC-MS/MS
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method using multiple mass transitions, rather than a single transition, allows
accurate quantitation of urinary cortisol in patients taking Fenofibrate.
Lassner A et al (2001)17 reported a sensitive HPLC method for the
estimation of Fenofibric acid (FA), which is an active form of Fenofibrate in serum.
An easy one-step extraction procedure was carried for the isolation of FA from
human serum samples by using a mixture of n-hexane and ethylacetate (90: 10 v/v).
The recovery was 84.8% of the total Fenofibric acid in serum. The Seperation was
done isocratically on RP-HPLC using acetonitrile and 0.02M phosphoric acid
(55:45) at a flow rate of 1.0 ml/min, UV detection was found at 287 nm.
Streel B et al (2001)18 reported a fully automated RP-HPLC with UV
detection method for the estimation of Fenofibrate in plasma, which involves the
solid-phase extraction (SPE) of the analyte from plasma on disposable extraction
cartridges (DECs). The cartridge was conditioned with methanol and pH-7.4
phosphate buffer first then filled with octadecyl silica and Sulindac (internal
standard) along with diluted plasma of 0.8ml volume was applied on the DEC. The
liquid chromatographic (LC) separation of the analytes was carried on a Nucleosil
RP8 stationary phase (5 micron), mobile phase used is a mixture of methanol and
0.04 M phosphoric acid (60:40 v/v) and UV detection was at 288 nm.
Lacroix PM et al (1998)19 reported an HPLC method for the estimation of
drug content, HPLC and NMR methods for related compounds in Fenofibrate raw
materials. By HPLC method 11 known and six unknown impurities were resolved
from the drug. Chromatographic separation was carried on Waters Symmetry ODS
column (100 x 4.6 mm, 3.5 micron), a mobile phase consisting of acetonitrile/water/
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trifluoroacetic acid 700/300/l (v/v/v) with a flow rate of 1.0 ml/min with UV
detection at 280 nm. An NMR method for related compounds was developed which
is suitable for 12 known and several unknown impurities, 400 MHz or greater NMR
is required.
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5.3. EXPERIMENTAL: METHOD DEVELOPMENT 5.3.1. MATERIALS AND METHODS
Materials
Fenofibrate and Nevirapine were obtained from CIPLA Pharmaceuticals,
Mumbai. HPLC grade acetonitrile, ethylacetate and methanol were purchased from
SD fine chemicals, Mumbai, India. Analytical Grade ammonium acetate was
purchased from SD fine chemicals, Mumbai, India. Pooled drug free expired human
plasma was donated by Red Cross Society, Warangal.
Instrumentation
The HPLC system consisted of Alliance Waters 2695 with dual λ
Absorbance UV detector. Separation was carried out on Inertsil C18 column
(4.6x250mmx5µm). A vortex-mixer (Remi), ultrasonic bath (Bransonic),
a model C-30 centrifuge (Remi) and a model cool safe 110-4/scan speed 32 were
used for sample preparation.
Chromatographic conditions
The HPLC system consisted of Alliance waters 2695 with dual λ Absorbance
UV detector. The wavelength of detection as set at 295nm. Separation was carried
out on Inertsil C18 column (4.6x250mmx5µm) using 60:40 v/v ammonium acetate
buffer and acetonitrile as mobile phase at a flow rate of 1.0 ml/min. The mobile
phase was filtered through nylon millipore (0.2µm) membrane filter, purchased from
pall life sciences, Mumbai and degassed with Ultrasonicator prior to use.
Chromatography was carried out at room temperature 250c .
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Preparation of Stock solutions
Stock solutions of Fenofibrate (500 µg/ml) and Nevirapine (1000 µg/ml)
internal standard were prepared in Methanol. Further dilutions were carried out in
60% acetonitrile.
Preparation of calibration standards
Calibration standards were prepared freshly by spiking drug free plasma with
Fenofibrate stock solution to give the concentrations of 0.3, 0.6, 1.2, 2.5, 5.0, 10.0
and 20.0µg/ml respectively; they were stored at -200c till the time of analysis.
Preparation of quality control standards
Quality control standards were prepared at three levels namely LQC, MQC
and HQC. These standards were prepared freshly by spiking drug free plasma with
Fenofibrate stock solution to give the concentrations of 1.0, 8.0 and 16.0µg/ml
respectively, they were stored at -200c till the time of analysis. Detailed procedure
for the preparation is shown in Table 46.
Sample preparation method
Drug free human plasma (500µl) was spiked with appropriate volume of
drug stock. To the above prepared sample, 50µl of Nevirapine (50µg/ml) was added
as an IS. The sample was vortex mixed for 4 min to assure complete mixing.
Analytes were extracted by adding with 3.0ml of ethyl acetate followed by vortex
mixing for 4 min and centrifugation at 2000 rpm/min in a cooling centrifuge for
15min at 40C. The organic phase containing analytes were separated and analytes
were obtained as dried residues after drying in lyophilizer. The analyte residue
obtained was reconstituted with 250µl of mobile phase and analysed using HPLC
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system according to parameters optimized. Detailed procedure for the preparation is
shown in Table 42. Calibration data is shown in Table-43, calculated concentrations
of calibrations standards and mean concentrations of calibration standards data were
showed in Tables 44 and 45.
5.4 EXPERIMENTAL: METHOD VALIDATION 5.4.1 VALIDATION PARAMETERS Specificity
A solution containing 0.3µg/ml Fenofibrate was injected onto the column
under optimized chromatographic conditions to show the separation of Fenofibrate
from the impurities from the plasma. The specificity of the method was checked for
the interference from plasma.
Linearity and Range
Six samples of each calibration standard were analysed. Wide range
calibration was determined by solutions containing 0.3µg/ml to 20.0µg/ml. The
spiked concentration and their respective peak area ratios with respective to internal
standards were subjected to least squares regression. After examining the percent
deviation, a proper model was chosen. The slope, Y-intercept, and coefficient of
determination (r2) were obtained from linear regression equation.
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
The limit of quantitation refers to the lowest amount of an analyte in a
sample that can be quantitatively determined with suitable precision and accuracy.
There are different approaches to determine the LOD and LOQ. Typically the
concentration level that generates a signal-to-noise (S/N) of 3.3 is regarded as the
LOD and the concentration level that generates S/N = 10 is regarded as the LOQ.
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Here LOD was calculated using the formula 3.3 times σ/s, similarly LOQ was
calculated using the formula 10 times σ/s where “σ” is the standard deviation of the
intercept obtained for calibration curve and “s” is the slope of the calibration curve.
Precision and accuracy
Intraday precision and accuracy was determined by analyzing quality control
standards (1.0, 8.0 and 16.0µg/ml) and LLOQC standards (0.15µg/ml) five times a
day randomly, interday precision and accuracy were determined from the analysis of
each quality control standards (1.0, 8.0 and 16.0µg/ml) and LLOQC standards
(0.3µg/ml) once on each of five different days.
Ruggedness
The ruggedness of the method was determined by analysing spiked control
samples of (n=6) medium concentrations i.e. 8.0µg/ml using two different columns.
Recovery studies
The percent mean recoveries were determined by measuring the responses of
the extracted plasma Quality control samples at HQC, MQC and LQC against
unextracted Quality control samples at HQC, MQC and LQC.
For calculation of recovery of the compound, spiked control samples were
prepared at low, medium and high concentrations i.e. 1.0, 8.0 and 16.0µg/ml
concentrations. The samples were processed as mentioned above and the
concentration of the compound was determined from the regression of the analytical
standard calibration curve. Recovery was calculated by comparing the observed
concentrations in spiked samples to that of respective unextracted samples.
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Stability studies
The stability of Fenofibrate was determined by measuring concentration
change in control samples overtime under set conditions.
Freeze-thaw stability study of Fenofibrate was carried out by subjecting
samples to three freeze and thaw cycles. Samples before study and after study were
analysed by developed method. Similarly Stock solution stability study of
Fenofibrate (Stability after ‘0’ hrs), Stock solution stability study (stability after
‘8’ hrs), Bench top stability study of Fenofibrate (Stability after ‘10’ hrs) and Inter
injection stability study of Fenofibrate were carried out by subjecting samples to
study conditions.
5.4.2 DATA ANALYSIS 20
Data analysis was done using MS-Excel package for calculating mean,
standard deviation and % relative standard deviation.
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5.5. RESULTS AND DISCUSSION
Under the chromatographic conditions employed, the sample showed sharp
peaks for drug and internal standard with good resolution. The retention time for the
Fenofibrate was found to be 6.672±0.05 minutes and the retention time for
Nevirapine (IS) was 5.213±0.03 minutes. . The results of validation parameters are
shown in Figure 31. The method developed was validated for specificity, accuracy,
precision, linearity, range and stability as per USFDA guidelines.
The specificity of the method was proven by the absence of peaks near the
retention time of the drug as well as the internal standard (Figure 32).
The calibration function was developed for Peak area ratio Vs Concentration
(in µg/ml) and it was linear over the concentration range of 0.3 to 20.0µg/ml. The
regression line equation for the analysis was y= 0.058x - 0.008 with a coefficient of
correlation (r2) = 0.999. The chromatogram of calibration standards and calibration
curve were shown in Figures 33 to 39.
The LOD is calculated using the formula 3.3 times σ/s, similarly LOQ is
calculated using the formula 10 times σ/s where “σ” is the standard deviation of the
intercept obtained for calibration curve and “s” is the slope of the calibration curve.
The calculated LOD and LOQ are shown in Table 47.
A system suitability test was carried out by injecting six aqueous mixtures of
Fenofibrate and Nevirapine (IS). The following parameters were observed for
repeated injections (n=6) of both Fenofibrate and Nevirapine (IS) (Table 48).
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The intraday and interday precision and accuracy of the method was found to
be 0.09 to 5.03% and 100.00 to 100.20% respectively for the quality control
samples. This is within the acceptance limits of precision is 15% and accuracy is 85
to 115% (Table 49). The limit of quantitation was found to be 0.3µg/ml at such
concentration the inter day precision was found to be 0.07 to 0.29 and the accuracy
was 100% to 100% respectively for the quality control samples, which are within
the acceptance limits of precision is 20% and accuracy is 80 to 120% (Table 50).
The results for ruggedness on different columns were shown in Table 52.
The percent mean recovery for Fenofibrate in LQC, MQC and HQC was
62.7%, 64.2 % and 65.3% respectively (Tables 53 to 55.).
Stability was assessed by comparing against the freshly thawed quality
control samples. The percent mean stability for HQC and LQC were 100.00% and
100.00% respectively, which is within the acceptance limits of 85 to 115%. Plasma
Quality control samples of Fenofibrate were found to be stable for at least one
month. The results were shown in the Table 56. Results for Stock solution stability
study (Stability after ‘0’ hrs), Stock solution stability study (Stability after ‘8’ hrs),
Bench top stability (Stability after ‘10’ hrs) and Inter injection stability study were
shown in Tables 57 to 60.
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Table 42: Preparation of Fenofibrate calibration standards in Plasma
Fenofibrate Concentration
(µg/ml) Drug stock
solution (µl)* Blank plasma (µl)
FFB CS1 0.3 12 988
FFB CS2 0.6 24 976
FFB CS3 1.25 50 950
FFB CS4 2.5 100 900
FFB CS5 5.0 200 800
FFB CS6 10.0 400 600
FFB CS7 20.0 800 200
* indicates drug stock solution from 10 µg/ml
Table 43: Calibration data of Fenofibrate
Concentration (µg/ml)
Peak Area Ratio of Peak Area Fenofibrate Internal Standard
0.3 5761 522497 0.011
0.6 11745 554621 0.021
1.25 25760 566244 0.045
2.5 78951 542818 0.145
5.0 158518 551221 0.287
10.0 323629 547925 0.59
20.0 625822 540980 1.156
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Table 44: Calculated concentrations of Fenofibrate calibration standards
Fenofibrate Actual
Concentration (µg/ml)
Calculated concentrations
1 2 3
FFB CS1 0.3125 0.307 0.317 0.332
FFB CS2 0.625 0.617 0.673 0.547
FFB CS3 1.25 1.242 1.255 1.234
FFB CS4 2.5 2.546 2.564 2.564
FFB CS5 5.0 4.973 4.813 5.067
FFB CS6 10.0 10.05 10.11 10.303
FFB CS7 20.0 20.297 19.404 20.146
Table 45: Mean concentrations of Fenofibrate calibration standards
Fenofibrate Concentration
(µg/ml) Mean of calculated
concentrations SD % RSD
FFB CS1 0.3125 0.3186 0.013 3.95
FFB CS2 0.625 0.6123 0.063 10.31
FFB CS3 1.25 1.243 0.011 0.85
FFB CS4 2.5 2.558 0.010 0.41
FFB CS5 5.0 4.951 0.128 2.59
FFB CS6 10.0 10.155 0.132 1.30
EPR CS7 20.0 19.949 0.478 2.40
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Table 46: Preparation of Fenofibrate quality control standards in Plasma
Fenofibrate Concentration (µg/ml)
Drug stock solution (µl)*
Blank plasma (µl)
FFB LQC 1.0 40 960
FFB MQC 8.0 320 680
FFB HQC 16.0 640 360
* indicates drug stock solution from 10 µg/ml
Table 47: Calibration curve parameters for Fenofibrate
1 2 3 Mean SD
Slope 0.0246 0.0255 0.0247 0.0249333 0.000493
Intercept 0.000322 0.00148 0.000448 0.00075 0.000635
r2 0.999 0.997 0.998 - -
LOD 0.084 µg/ml - - - -
LOQ 0.254 µg/ml - - - -
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Table 48: HPLC System suitability parameter (Precision) for Fenofibrate and Nevirapine (IS)
Name Replicates RT Peak area
Fenofibrate (n=6)
1 7.059 673863
2 7.036 648921
3 7.019 669539
4 7.047 659253
5 7.051 670216
6 7.039 668539
Mean 7.041 665055.16
SD 0.0139 9278.25
% RSD 0.19 1.39
Internal Standard (n=6)
1 5.271 29251
2 5.239 27639
3 5.277 25453
4 5.204 26402
5 5.253 28493
6 5.228 29026
Mean 5.245 27710.66
SD 0.0274 1518.52
% RSD 0.5 5.4
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Table 49: Intra-day accuracy and precision for Fenofibrate
Fenofibrate QC ID LQC MQC HQC
Actual conc.(µg/ml) 1.0 8.0 16.0
Intraday
1 1.062 8.527 17.05
2 1.076 8.447 17.09
3 1.068 8.975 17.075
4 1.073 8.046 17.069
5 1.073 9.131 17.087
Mean 1.070 8.625 17.074
± SD 0.0055 0.4344 0.0160
% RSD 0.5142 5.0372 0.0938
% Accuracy 100.00 100.20 100.00
Table 50: Inter-day accuracy and precision for Fenofibrate
Fenofibrate QC ID LQC MQC HQC
Actual conc.(µg/ml) 1.0 8.0 16.0
Interday
1 1.049 8.176 16.495
2 1.055 8.177 16.481
3 1.05 8.184 16.514
4 1.055 8.181 16.493
5 1.049 8.191 16.508
Mean 1.051 8.181 16.498
± SD 0.0031 0.0060 0.0130
% RSD 0.2976 0.0740 0.0789
% Accuracy 100.00 100.00 100.00
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Table 51: Precision & Accuracy of LLOQC Standard
QC ID LLOQC Actual conc.(µg/ml) 0.3125
1 0.307
2 0.323
3 0.319
4 0.317
5 0.332
Mean 0.3196
±SD 0.0090
%RSD 2.8471
% Accuracy 100.06
Table 52: Ruggedness of the method developed for
Fenofibrate (on different columns)
MQC
Replicates
Column 1
(area ratio)
Column 2
(area ratio)
1 0.869 0.886
2 0.873 0.886
3 0.878 0.864
4 0.884 0.882
5 0.862 0.842
6 0.852 0.855
Mean 0.870 0.869
± SD 0.011 0.018
% RSD 1.32 2.12
% Accuracy 100.06
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Table 53: Recovery study for Fenofibrate from human plasma - LQC
Fenofibrate
Replicates
LQC
Unextracted
(area ratio)
Extracted
(area ratio)
%Recovery
1 0.099 0.062 62.63
2 0.098 0.063 64.29
3 0.098 0.064 65.31
4 0.098 0.058 59.18
5 0.098 0.061 62.24
6 0.099 0.062 62.63
Mean 0.098 0.062 62.712
±SD 0.001 0.002 2.092
%RSD 0.53 3.35 3.34
Table 54: Recovery study for Fenofibrate from human plasma -MQC
Fenofibrate
Replicates
MQC
Unextracted
(area ratio)
Extracted
(area ratio)
%Recovery
1 0.867 0.554 63.90
2 0.867 0.556 64.13
3 0.867 0.549 63.32
4 0.866 0.569 65.70
5 0.867 0.574 66.21
6 0.867 0.542 62.51
Mean 0.867 0.557 64.296
±SD 0.0004 0.012 1.410
%RSD 0.05 2.17 2.19
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Table 55: Recovery study for Fenofibrate from human plasma - HQC
Fenofibrate
Replicates
HQC
Unextracted
(area ratio)
Extracted
(area ratio)
%Recovery
1 1.573 1.048 66.62
2 1.572 1.057 67.24
3 1.574 0.993 63.09
4 1.572 0.992 63.10
5 1.569 1.082 68.96
6 1.566 0.992 63.35
Mean 1.571 1.027 65.394
±SD 0.003 0.040 2.546
%RSD 0.19 3.89 3.89
Table 56: Freeze-thaw Stability of Quality Control Standards
Stability after 3 freeze thaw cycles
Fenofibrate LQC HQC
Actual conc.(µg/ml) 1.0 16.0
1 0.975 17.473
2 0.983 17.516
3 0.983 17.657
4 0.956 17.48
5 0.982 17.473
6 0.98 17.628
Mean 0.976 17.537
± SD 0.0104 0.0831
% RSD 1.0735 0.4740
% Accuracy 100.00 100.00
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Table 57: Stock solution stability study of Fenofibrate (Stability at ‘0’ hrs)
Replicates SSS(0hrs) LQC SSS(0hrs) HQC
1 0.267 4.696
2 0.265 4.649
3 0.272 4.724
4 0.276 4.745
5 0.268 4.73
6 0.271 4.724
Mean 0.270 4.711
± SD 0.004 0.034
% RSD 1.47 0.73
Table 58: Stock solution stability study of Fenofibrate (Stability after ‘8’ hrs)
Replicates SSS(8hrs) LQC SSS(8hrs) HQC
1 0.262 4.412
2 0.265 4.457
3 0.263 4.459
4 0.266 4.449
5 0.261 4.454
6 0.262 4.501
Mean 0.263 4.455
± SD 0.002 0.028
% RSD 0.74 0.64
% Accuracy 97.53 94.57
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Table 59: Bench top stability study of Fenofibrate (Stability after 10 Hours)
Time (h) 10.00(h)BTS
Fenofibrate LQC HQC
Actual conc.(µg/ml) 1.0 16.0
1 0.983 17.404
2 0.978 17.439
3 0.985 17.58
4 0.99 17.342
5 0.977 17.338
6 0.981 17.348
Mean 0.982 17.408
± SD 0.0048 0.0931
% RSD 0.4889 0.5352
% Accuracy 100.00 100.00
Table 60: Inter injection stability study of Fenofibrate
Fenofibrate LQC HQC
Actual conc.(µg/ml) 1.0 16.0
1 1.091 17.394
2 1.09 17.347
3 1.095 17.468
4 1.094 17.298
5 1.089 17.295
6 1.093 17.291
Mean 1.092 17.348
± SD 0.0023 0.0708
% RSD 0.2167 0.4081
% Accuracy 100.00 100.00
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CHROMATOGRAMS OF DEVELOPED METHOD
Figure 31: Chromatogram showing Retention a time of Aqueous Mixture consists of Fenofibrate (5µg/ml) and Nevirapine (5µg/ml).
Figure 32: Chromatogram of Blank plasma sample showing no interference at the RT of Fenofibrate and Nevirapine
160
Figure 33: Chromatogram of Fenofibrate calibration standard-1(FFB CS1)
Figure 34: Chromatogram of Fenofibrate calibration standard-2(FFB CS2)
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Figure 35: Chromatogram of Fenofibrate calibration standard-3(FFB CS3)
Figure 36: Chromatogram of Fenofibrate calibration standard-4(FFB CS4)
162
Figure 37: Chromatogram of Fenofibrate calibration standard-5(FFB CS5)
Figure 38: Chromatogram of Fenofibrate calibration standard-6(FFB CS6)
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Figure 39: Chromatogram of Fenofibrate calibration standard-7(FFB CS7)
Figure 40: Calibration curve of Spiked concentrations (FEB CS1-CS7)
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Figure 41: Chromatogram of Fenofibrate Low Quality Control (FFB LQC)
Figure 42: Chromatogram of Fenofibrate Middle Quality Control (FFB MQC)
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Figure 43: Chromatogram of Fenofibrate High Quality Control (FFB HQC)
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5.6. SUMMARY
A Simple, rapid, selective and sensitive HPLC method was developed and
validated for the determination of Fenofibrate from human plasma. The drug was
extracted with ethyl acetate. Fenofibrate was measured in plasma using a validated a
HPLC method with UV detector at 295nm chromatographic peaks were separated
on 5µm Intensil, C18 column (4.6x250mmx5µm) using 60:40 v/v 20mM ammonium
acetate buffer with initial pH and acetonitrile as mobile phase at a flow rate of 1.0
ml/min. The chromatograms showed good resolution and no interference from
plasma. The retention time of Fenofibrate and Nevirapine (IS) were approximately
6.6±0.05 min and 5.2±0.03 min respectively. The mean recovery from human
plasma was found to be above 62%. The method was linear over the concentration
range of 0.3 to 20.0µg/ml with a coefficient of correlation (r2) 0.999. Both intraday
and interday accuracy and precision data showed good reproducibility. This method
was successfully applied to pharmacokinetic studies.
5.7 CONCLUSION
Various methods reported in literature were studied. In the present study a
simple, rapid, specific, rugged, accurate, precise and stable method was developed
for the estimation of Fenofibrate in human plasma. The calibration curve developed
is y= 0.058x - 0.008µg/ml with r2=0.999. The percent mean recovery for
Fenofibrate in LQC, MQC and HQC was 62.7%, 64.2 % and 65.3% respectively.
The method is accurate, precise and rugged with % RSD < 15% and 20% when
tested at MQC, HQC and LQC levels respectively. The stability was assessed at
different levels. The results of the Freeze-thaw stability, Bench top stability and
Inter injection stability studies showed that the compound under analysis is stable
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under test conditions. Hence the method developed can be used for estimation of
Fenofibrate present in human plasma.
168
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